ER "quality control" (ERQC) is a fundamental and conserved cellular process that prevents the exit of misfolded secretory and membrane proteins from the ER. ERQC consists of two sequential processes: 1) the unfolded protein response (UPR), which refers to the transcriptional upregulation of genes such as chaperones that enable the cell to cope with misfolded proteins, followed by 2) ER-associated degradation (ERAD), whereby misfolded ER-retained proteins are degraded by the ubiquitin-proteasome system. Recent studies suggest that for membrane proteins there are two classes of ERAD substrates, based on the topological location of their misfolded lesion, either luminal (L) or cytosolic (C). In the present project, we propose an extension of this view, namely that cells employ two mechanistically distinct branches of ER quality control: ERQC-L (comprising UPR-L and ERAD-L) and ERQC-C (comprising UPR-C and ERAD-C), to cope with substrates whose domains are luminal or cytosolic, respectively. The long-term goal of this project is to identify and mechanistically dissect the components and workings of the ERQC-C pathway in Saccharomyces cerevisiae, and determine how ERQC-C differs from ERQC-L. Evidence for distinct branches of ERQC is based on our studies of mutant forms of the yeast ATP-binding cassette (ABC) transporters Ste6p and Ycflp, that are subject to ERQC. Using these as model ERQC-C substrates, we have made significant advances in this project that include defining a prominent ER compartment (the ERAC) as a marker for UPR-C, defining differences in machinery between the ERAD-C and ERAD-L, and gaining an initial glimpse into differences in the transcriptional induction profiles of UPR-C and UPR-L. These findings set the stage for the present proposal. Here, we will apply traditional and high-throughput yeast genetic, molecular, and cell biological methodologies to accomplish the following aims: 1) To elucidate the circuitry of the UPR-C signaling pathway by defining the key regulators, upregulated genes, and cytoprotective mechanisms evoked by a UPR-C stress; 2) To define the machinery, steps, and mechanism of the ERAD-C pathway by probing the substrate specificity of E3 ubiquitin ligases and identifying novel ERAD components; and 3) To further develop MRP proteins as model ERQC substrates, in particular to gain new insights into the relationship between ERQC and ER exit. Our studies are expected to shed light on a diverse array of membrane protein trafficking diseases, best exemplified by cystic fibrosis, which most commonly results from the ER-retention and degradation of CFTR-deltaF508.